Silicon improves ion homeostasis and growth of liquorice under salt stress by reducing plant Na+ uptake

Silicon (Si) effectively alleviates the effects of salt stress in plants and can enhance salt tolerance in liquorice. However, the mechanisms by which Si improved salt tolerance in liquorice and the effects of foliar application of Si on different liquorice species under salt stress are not fully understood. We investigated the effects of foliar application of Si on the growth, physiological and biochemical characteristics, and ion balance of two liquorice species, Glycyrrhiza uralensis and G. inflata. High salt stress resulted in the accumulation of a large amount of Na+, decreased photosynthetic pigment concentrations, perturbed ion homeostasis, and eventually inhibited both liquorice species growth. These effects were more pronounced in G. uralensis, as G. inflata is more salt tolerant than G. uralensis. Foliar application of Si effectively reduced the decomposition of photosynthetic pigments and improved gas exchange parameters, thereby promoting photosynthesis. It also effectively inhibited lipid peroxidation and leaf electrolyte leakage and enhanced osmotic adjustment of the plants. Furthermore, Si application increased the K+ concentration and reduced Na+ absorption, transport, and accumulation in the plants. The protective effects of Si were more pronounced in G. uralensis than in G. inflata. In conclusion, Si reduces Na+ absorption, improves ion balance, and alleviates the negative effects of salt stress in the two liquorice species studied, but the effect is species dependent. These findings may help to develop novel strategies for protecting liquorice plants against salt stress and provide a theoretical basis for the evaluation of salt tolerance and the scientific cultivation of liquorice.


Experimental design
The treatments were arranged in a completely randomised block design with three replicates. To explore the effects of leaf application of Si on the two liquorice species under different salt concentrations, based on the preliminary results, the following treatments were chosen for detailed analysis: (1) control (CK); (2) control + 3 mM Si (CK + Si); (3) 6 g kg -1 NaCl (6S); (4) 6 g kg -1 NaCl + 3 mM Si (6S + Si); (5) 12 g kg -1 NaCl (12S); and (6) 12 g kg -1 NaCl + 3 mM Si (12S + Si). Si was applied as a foliar spray in the form of K 2 SiO 3 in Si treatments. To avoid the influence of osmotic pressure caused by K + when K 2 SiO 3 was added, 6 mM KCl was added to the control and treatment without Si, respectively. When the content of Clwas low, its influence on plant growth could be ignored 37 . www.nature.com/scientificreports/ Ten seeds of liquorice were sown per plastic pot (23.5 × 16 × 18 cm 3 ) containing 5 kg of sandy soil. After the emergence of 2 or 3 true leaves (15 d after sowing), 6 robust and disease-free liquorice seedlings of the same height and growth were retained in each pot and watered every day. NaCl and Si treatments were conducted simultaneously 4 weeks after sowing. Except for the treatments, other management measures were consistent with those of local field management practices. After 100 days of treatment, the growth parameters and physiological and biochemical characteristics of each liquorice species were determined. Determination of growth parameters. Plant height was measured using a tape measure, and stem thickness was measured using a Vernier calliper. The leaves, stems, and roots of liquorice were washed and placed in an oven at 105 °C for 30 min, dried at 75 °C for 48 h, and weighed.
Determination of gas exchange attributes. Net photosynthesis rate (P n ), transpiration rate (T r ), stomatal conductance (g s ), and intercellular CO 2 concentration (C i ) were recorded between 9:00 am and 12:00 am using a Li-6400 photosynthesis instrument (Li-COR, Lincoln, NE, USA). Three similarly sized healthy and fully expanded leaves from the top of the stem from plants under each treatment were analysed at a leaf temperature of 28 °C, irradiance of 1200 μmol m -2 s -1 , and CO 2 concentration of 400 μmol mol -1 .
Determination of photosynthetic pigments. The veins of fresh leaves were removed, cut into pieces, and weighed. Then, 10 mL of 80% (v/v) acetone was added to 0.5 g of the plant material and extracted in the dark until the leaves were colourless. Next, the supernatant was obtained after centrifugation at 4000 r min −1 for 10 min. Then we added 4 mL of 80% acetone to 1 mL of supernatant, and the absorbance values of the extracts were determined at 470 nm, 646 nm, and 663 nm using a Shimadzu UV-1900 spectrophotometer (Kyoto, Japan). The concentrations of Chl a, Chl b, total Chl, and carotenoids were calculated according to Lichtenthaler and Wellburn 38 .
Determination of Na + and K + concentrations, transfer, and absorption. Dry plant samples (0.1 g; various portions, as specified) were digested in a mixture of nitric acid and perchloric acid (volume ratio 2:1). The leaf and root concentrations of Na + and K + were measured using a flame photometer (FP640, Shanghai Precision Scientific Instrument Co., Ltd., Shanghai, China). Sodium uptake at the liquorice root surface and ion (Na + and K + ) translocation from the root to shoot were calculated using the methods described by Yan et al. 39 and Ali et al. 5 , respectively.
Determination of soluble sugar, soluble protein, and proline contents. For soluble sugar determination, fresh leaves (0.2 g) were ground into a homogenate in 6 mL of distilled water and then incubated in a water bath at 100 °C for 20 min. After cooling, the samples were centrifuged at 3000 r min -1 for 10 min. The extract (1.0 mL) was then mixed with 5 mL of anthrone reagent, and the absorbance value at 620 nm was measured using a Shimadzu UV-1900 spectrophotometer (Kyoto, Japan). The soluble sugar content was then calculated using a standard curve of sucrose 40 .
Soluble protein was determined using the Coomassie Brilliant Blue G-250 method 41 . Fresh leaves (0.2 g) were added to phosphate buffer solution (pH 7.0), ground into a homogenate, and centrifuged at 5000 r min -1 for 10 min. Coomassie Brilliant Blue G-250 reagent was added to 1 mL of the supernatant and the absorbance was read at 595 nm. The protein content was calculated using a standard curve of bovine serum albumin 40 .
For proline content determinations, fresh liquorice leaves (0.5 g) were placed in 5 mL of 3% sulfosalicylic acid solution and centrifuged at 5000 r min -1 for 10 min. The supernatant (2 mL) was added to 2 mL of glacial acetic acid and 2 mL of acidic ninhydrin reagent, and the mixture was heated in a boiling water bath for 30 min. After cooling, 4 mL of toluene was added, shaken for 30 s, and centrifuged at 5000 r min -1 for 10 min. Using toluene as a blank control, sample absorbance was measured at 520 nm by using a Shimadzu UV-1900 spectrophotometer (Kyoto, Japan), and the proline content was calculated using a standard curve by Li 40 . Determination of lipid peroxidation and LEL. Lipid peroxidation was determined by measuring the concentration of malondialdehyde (MDA). Liquorice leaves (0.5 g) were homogenised in 5 mL of 0.1% (w/v) trichloroacetic acid solution. After centrifugation at 10,000 r min -1 for 10 min, the supernatant was mixed with 0.5% (w/v) trichloroacetic acid solution and incubated in a water bath at 100 °C for 2 min. The samples were then centrifuged at 10,000 r min -1 for 10 min. The absorbance of the sample supernatant at 600 nm, 532 nm, and 450 nm was determined using a Shimadzu UV-1900 spectrophotometer (Kyoto, Japan), and the MDA content was calculated as described by Li 40 .
To determine the LEL, fresh liquorice leaves were cleaned, cut into 2 cm pieces, and placed in a test tube containing 10 mL of distilled water. The samples were shaken on an oscillating table at 25 °C for 24 h to determine electrical conductivity (EC 1 ). The test tube was then placed in a water bath at 100 °C for 30 min to determine electrical conductivity (EC 2 ) 42 . LEL was calculated using the following formula: LEL (%) = EC 1 /EC 2 × 100%.

Determination of antioxidant enzyme activities.
Fresh leaves (0.5 g) were added to phosphate buffer solution (pH 7.0), ground into a homogenate, and centrifuged at 10,000 r min -1 for 10 min. The supernatant was diluted to 25 mL using the same buffer solution. The samples were then stored in an icebox for determination of SOD and CAT activities. All operations were performed at 0-4 °C. SOD activity was determined using the nitrogen blue tetrazole method 40 , and CAT activity was determined using the colorimetric method 43  www.nature.com/scientificreports/ Statistical analysis. Analysis of variance was used to test the effect of different treatments on each index in the same liquorice species (P < 0.05), and Duncan's multiple comparisons test was used to determine significant differences between different treatments of the same liquorice. Meanwhile, data were checked for normality and the homogeneity of variances and the data of SOD and intercellular CO 2 concentration were transformed with natural logarithm to correct deviations from these assumptions, when needed. The overall data were analysed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA).

Compliance statement for experimental materials. Liquorice is a widely distributed species in
China. Xinjiang is the main producing area of liquorice in China. Seed of G. uralensis was collected from Wenquan County (Xinjiang, China; 80°57E, 44°58 N), and that of G. inflata was collected from Korla (Xinjiang, China; 86°12E, 41°69 N). A pot experiment was performed on the experimental field of the College of Life Science (Shihezi University, China; 86°06E, 44°30 N). Therefore, all operations comply with relevant institutional, national, and international guidelines and legislation.

Results
Plant growth. Salt stress significantly inhibited the growth of the two liquorice species. The inhibitory effect on plant growth was more pronounced in G. uralensis than in G. inflate. Under the 12S treatment, where 12 g kg -1 NaCl was used, both species displayed significant reductions in plant height, root dry weight, and shoot dry weight compared to their respective controls. However, between the two species, G. inflata showed better plant height, root dry weight, and shoot dry weight by 57%, 39%, and 42%, respectively, than G. uralensis (Table 1). Nevertheless, this marked inhibitory effect in G. uralensis was alleviated by Si (Table 1). Under the 12S + Si treatment, plant height, root dry weight, and shoot dry weight of G. uralensis increased by 74%, 81%, and 74%, respectively, compared to the corresponding values without Si. However, in the case of Si treatment of G. inflata, these indicators increased by 19%, 48%, and 49%, respectively compared to those of the control. The foliar application of Si resulted in a significant increase in growth characteristics of both the species. The growth enhancement was markedly higher in G. uralensis than G. inflata.

Gas exchange attributes. Salt treatment (6S and 12S) showed a reduction in gas exchange attributes with
increasing NaCl concentrations ( Fig. 1). However, in the case of 12S salt treatment, G. uralensis showed a 41% increase in C i compared to that in the untreated control (Fig. 1d). The various gas exchange parameters analysed indicated an overall enhancement with respect to Si treatment in both the species, irrespective of the NaCl concentration. A similar result was observed for the CK + Si plants that showed a marked increase in all the gas exchange parameters with Si treatment. However, in the 12S + Si treatment, G. uralensis showed a 16% reduction in the C i levels (Fig. 1d). Between the two species, G. uralensis showed greater increase in gas exchange parameters in the 6S + Si treatment. G. uralensis showed a 78% increase (Fig. 1a) in P n compared to that of G. inflata, which only showed a 29% increase in P n . In G. uralensis, g s increased by 59% (Fig. 1b) compared to a 15% increase in G. inflata. Furthermore, G. uralensis showed a 55% increase (Fig. 1c) in T r and a 33% increase in C i (Fig. 1d), whereas G. inflata showed only a 15% increase for both the parameters.

Photosynthetic pigments concentrations.
Salt treatment significantly reduced the photosynthetic pigments concentrations in both the liquorice species compared to those in CK (Fig. 2). However, Si treatment (CK + Si, 6S + Si, and 12S + Si) resulted in a marked increase in chlorophylls and carotenoids concentrations in both control and salt-treated plants (Fig. 2a-d). In the 12S + Si treatment, G. inflata showed a marginal 5% increase of Chl b when compared to G. uralensis, which showed a 33% increase of Chl b (Fig. 2b). www.nature.com/scientificreports/ Accumulation of Na + and K + in the root and leaf. The accumulation of Na + in the root and leaf of the two liquorice species increased significantly with increasing salt concentrations, with a simultaneous significant decrease in the accumulation of K + (Fig. 3). G. uralensis showed a significant increase in the accumulation of Na + in the leaf tissue compared to that of G. inflata. The CK, 6S, and 12S salt treatments resulted in a 49%, 35%, and 57% increase in Na + accumulation, respectively, in G. uralensis (Fig. 3b). Si application caused a significant reduction in the accumulation of Na + in the root and leaf tissues of both the species in the 6S as well as 12S salt treatments (Fig. 3a,b). The application of Si with the 6S treatment resulted in a 27% reduction in Na + accumulation in the leaves of G. uralensis (Fig. 3b) and a subsequent 28% increase in K + accumulation (Fig. 3d).
Ion translocation and uptake. Salt treatment significantly increased Na + transport (root-leaf) and Na + uptake on the root surface and decreased the K + /Na + ratio in both the liquorice species (Fig. 4). K + transport and K + /Na + ratio in G. inflata were 50% and 71% higher than those in G. uralensis under the 6S treatment, respectively, while Na + transport and Na + uptake on the root surface of G. uralensis were 35% and 93% higher than those of G. inflata, respectively. The application of Si decreased the Na + transport and increased the K + transport and K + /Na + ratio, thereby significantly affecting the uptake of Na + on the root surface under 12S treatment. 12S + Si treatment reduced the uptake of Na + by 49% in G. uralensis and 46% in G. inflata.
Soluble sugar, soluble protein, and proline contents. Salt treatment resulted in a significant increase in the accumulation of soluble sugars in both the species (Fig. 5a) with the maximum accumulation in the 6S samples. In the 6S samples, soluble sugars of G. uralensis and G. inflata were 101% and 61% higher than those in the CK samples, respectively. Si application also resulted in 18% and 22% increase of sugars accumulation in the 6S + Si samples of both the two species. The accumulation of soluble protein (Fig. 5b) was the maximum in the 6S samples with a 97% increase in G. uralensis and a 38% increase in G. inflata. Si application resulted in an increase in soluble protein concentration in both the species. Compare with the CK samples, accumulation of proline (Fig. 5c) was the highest in the 6S samples for G. uralensis by 125%, whereas for G. inflata, the maximum accumulation of proline was obtained in the 12S samples by 242%. In addition, Si application resulted in an increase in proline concentrations in both the species across all salt treatments. Especially at the 6S + Si treatment, where  Lipid peroxidation and LEL. The accumulation of MDA and LEL significantly increased with increase in salt concentrations in both the liquorice species. The changes were more pronounced in G. uralensis than in G. inflata (Fig. 6). Si application resulted in a significant reduction in the MDA levels and LEL in both the species.
In the 12S + Si treatment, Si application reduced the accumulation of MDA by 33% in G. uralensis and 19% in G. inflata. Similarly, Si application significantly reduced LEL by 16% in G. uralensis and 22% in G. inflata.

Antioxidant enzyme activities. SOD and CAT activities of the two species were both increased at 6S and
12S treatments compared with the CK, which for G. uralensis showed the maximum activity of 28% and 128% in the 6S treatment, whereas those for G. inflata were 25% and 166% in the 12S treatment (Fig. 7). However, Si application resulted in increased SOD and CAT activities across all salt treatments in both the species. The exogenous application of Si under 6S treatment improved the activities of SOD and CAT in G. uralensis by 20% and 59%, while the increment was about 10% and 61% respectively in G. inflata, as compared with 6S treatment alone.
Correlation analysis. Pearson's correlation analysis was performed to monitor the differences in plant growth, physiology, and biochemical attributes along with ion homeostasis of G. uralensis and G. inflata (Fig. 8).
The concentration of Na + in liquorice roots showed a positive correlation with Na + in the leaves, Na + absorption on the root surface, and oxidative stress indexes MDA and LEL. Na + in liquorice roots showed a negative correlation with growth indexes, gaseous exchange attributes (except Ci for G. uralensis), Chl content, K + in the roots and leaves, and K + /Na + ratio.

Discussion
Salinity stress is one of the main adverse environmental conditions encountered by plants 1 . The detrimental effects of salinity include ion toxicity and osmotic stress, which cause growth inhibition, yield reduction, and may eventually lead to plant death 44 . Several studies indicate 45,46 that Si can be used to effectively alleviate the harmful effects of salt stress on plants and to promote plant growth in saline environments.
In the current study, we analysed the growth indicators, gas exchange parameters, Chl content, ion balance, osmotic regulators, membrane damage indicators, and antioxidant enzyme activity in the liquorice species G. uralensis and G. inflata. Although several studies have investigated the effect of Si on the growth of liquorice under salt stress [33][34][35]37,47 , this study demonstrates that exogenous application of Si by foliar spray enhances salt tolerance of G. uralensis and G. inflata.
Both medium-salt (6S) and high-salt (12S) treatments significantly reduced the biomass of the two liquorice species, with the lowest values obtained at high salt concentrations. Under the same salt treatment, the growth of G. inflata was better than that of G. uralensis, indicating that G. inflata is more salt tolerant than G. uralensis. However, the effects are evidently mitigated by foliar application of Si which resulted in increased biomass in both the species (Table 1).
We studied the gas exchange attributes of G. uralensis and G. inflata by analysing the P n , T r , g s , and C i in both the liquorice species. Foliar application of Si alleviated the negative effects of salt stress on all these gas exchange parameters in the two liquorice species (Fig. 1). Under high salt stress, we observed a significant increase in the C i levels in G. uralensis (Fig. 1d). This was an unexpected result; however, it is possible that the stomatal protection system is damaged when the salt concentration exceeds the tolerance limit of the species and can be attributed to the involvement of non-stomatal factors that play a major role in photosynthesis 48 . In addition, under high salt stress, the P n , T r , and g s of the two liquorice species were also significantly reduced. However, all of these negative effects were ameliorated by the foliar application of Si in both the species, further establishing the role of Si in ameliorating the effects of salt treatment.
Consistent with the findings for mung bean 9 and cucumber 19 , salt stress reduced photosynthetic pigments concentrations in the leaves of the two liquorice species (Fig. 2). However, foliar application of Si significantly increased chlorophylls and carotenoids concentrations in plant leaves under salt stress. These observations suggest that Si promotes the biosynthesis of photosynthetic pigments under salt stress. This may be attributed to www.nature.com/scientificreports/ the notion that Si alleviates the damage to chloroplast under salt stress 34 , enhances Rubisco protein expression, and promotes the synthesis of photosynthetic pigments 45 . The negative effects of salt stress such as damage to plant cells and the decrease in photosynthesis rate are mainly caused by excessive absorption and accumulation of Na +6,18 . When subjected to high salt stress, it is possible that the ability of the root system of G. uralensis to intercept Na + is compromised, which led to the high accumulation of Na + in the root and leaf tissues (Fig. 3). This in turn led to the disturbance in the synthesis of photosynthetic pigments 34 , a decrease in the net photosynthetic rate, and a significant reduction of plant biomass (Figs. 1a, 2, and Table 1). In high-salt environments, the accumulation of Na + increases in the roots, and the transport of Na + is enhanced towards the shoot, thus, increasing Na + accumulation in the leaves, which damages the mesophyll cells 49 . When the Na + concentration in the leaf exceeds 1.3 mg g -1 , the chloroplast structure is damaged, Chl degradation is accelerated, and photosynthesis is inhibited 50 . Our results indicate that the transport and absorption of Na + on the root surface were higher in G. uralensis than in G. inflata under medium-salinity conditions. This indicates that the ability of G. inflata roots to intercept Na + is greater than that of G. uralensis, which might account for the relatively high salt tolerance of G. inflata.
K + is the key regulator of cell homeostasis 8 and plays an important role in inducing cell elongation, maintaining osmotic regulation in plants, and promoting photosynthesis 8,51,52 . Therefore, excessive Na + levels often lead to K + deficiency. Al-Huqail et al. 45 reported that high salt concentrations significantly increase the Na + content in maize and greatly reduce the K + content, resulting in an increased Na + /K + ratio and plant growth inhibition. In G. uralensis, compared with the application of Si in the soil, foliar spray alone effectively increased K + levels and reduced Na + levels 35 . This may be associated with the Si-induced upregulation of genes involved in potassium uptake (OsAKT1 and OsSHAK1) and xylem load (OsSKOR) 53 , which promotes the increase of K + transport and increases the H + -ATPase activity, forming a mechanical barrier to reduce Na + transport 54 . Therefore, we believe that the capacity of Si to enhance K + -selective transport and increase the K + /Na + ratio might be the main mechanisms to improve plant growth and productivity under salt stress, which is in agreement with the findings of previous studies 5, 52 . In addition, Si not only reduced the Na + transfer and damage to the shoot under salt stress but also decreased the Na + absorption by the root in the two liquorice species (Fig. 3), which is consistent with previous findings in rice 17 and wheat 6 . There are at least two possible explanations: (1) Si deposition on the root cell wall enhances the mechanical strength of the cell wall, thus, reducing Na + absorption 13 ; and (2) Si decreases Na + accumulation in the root apex and cortex by upregulating the expression of ZmSOS1 and ZmSOS2 www.nature.com/scientificreports/ transporters 55 . Considering both scenarios, we could say that the reduction of Na + uptake and transport may well be a potential mechanism of Si-mediated enhancement of salt tolerance in liquorice.
In high-salt environments, plants accumulate high levels of Na + . This leads to excessive accumulation of ROS, perturbing the balance between their production and elimination, and resulting in cellular oxidative damage 9 . MDA and LEL are the main indexes used to evaluate the severity of oxidative cell damage 17 . MDA is an oxidation  www.nature.com/scientificreports/ product of membrane lipids and accumulates when plants are subjected to oxidative stress. Na + accumulation results in the production of high levels of ROS, which destroy the cell membrane structure, leading to an increased MDA content and LEL. For instance, salt-induced oxidative damage leads to the rupturing of the plasma membrane in maize, leading to lipid peroxidation 5 . In this study, we showed that both medium and high-salt treatments increased the MDA content and LEL in the two liquorice species, especially in G. uralensis (Fig. 6), indicating major damage of the cell membrane in these liquorice species. Of the two species, the damage was more pronounced in G. uralensis. However, the MDA content and LEL in the two liquorice species decreased after foliar spraying with Si. These observations suggest that Si may counteract the membrane damage caused by salt stress in liquorice. In addition, Si can alleviate reactive oxygen damage by maintaining membrane integrity and activating the antioxidant defence system. In this study, Si enhanced SOD and CAT activities in the two liquorice species under salt stress (Fig. 7), which is consistent with the observations in mung bean 9 and mustard 56 .  , Na-T (Na + translocation), K-T (K + translocation), K/Na ratio (K + /Na + ratio), Na uptake (Na + uptake). www.nature.com/scientificreports/ This effect may be related to the upregulation of genes encoding antioxidant enzymes 57 . We also observed that Si application enhanced the SOD and CAT activities to a greater extent in G. uralensis than in G. inflata. Based on the observations in this study, we could say that the regulation of the plant antioxidant system by Si upon salt stress is different for the two different liquorice species. The alleviating effect of Si described above was not only associated with the increased antioxidant enzyme activity but also with the accumulation of osmotic regulators in plants 45 . Accordingly, upon salt stress, plants produce and accumulate compatible organic solutes as a means of osmotic adjustment, to maintain the normal physiological and biochemical characteristics of intracellular water and to impede the damage to the cell membrane 48,58 . We demonstrated in this study that under high-salt stress, the soluble sugar, soluble protein, and proline contents in the leaf of G. uralensis were increased (Fig. 5), indicating that high-salt stress greatly affected the ability of G. uralensis to respond to the adverse effects of high-salt. Proline is a key osmoprotectant and can reduce the damage caused by ROS, reduce lipid peroxidation, and protect protein and membrane structures 48 . In the current study, the application of Si reduced the proline content in liquorice, which was consistent with the findings in sunflower 11 and pelargonium 24 . In the presence of Si, proline was degraded and used as a source of carbon and nitrogen in plants recovering from stress, as well as a membrane stabiliser and a free-radical scavenger to reduce lipid peroxidation and LEL 16,20 . Therefore, reduction of membrane damage may be another mechanism whereby Si improves the salt tolerance of plants. However, the mechanism for obtaining Si to improve plant salt tolerance is a complex process. In addition to many physiological and biochemical aspects, further research is required on protein and gene expression analysis.

Conclusions
High salinity greatly inhibited the growth and development of the two liquorice species studied. Under salt stress, the morphological characteristics (plant height, stem diameter, and biomass), photosynthetic characteristics (gas exchange parameters and photosynthetic pigments concentrations), antioxidant enzyme activities (SOD and CAT), ion homeostasis (K + and Na + transport, and K + /Na + ratio), and osmotic adjustment (e.g. proline) in G. inflata were better than those in G. uralensis. These observations indicate that G. inflata is more salt tolerant than G. uralensis. Foliar application of Si effectively reduced the absorption of Na + , improved ion balance, alleviated membrane damage, and promoted the growth of the two liquorice species. Furthermore, the response of G. uralensis to Si was more pronounced than that of G. inflata, indicating that the protective effect of Si is different for different liquorice species. This study provides a theoretical basis for the evaluation of salt tolerance in and scientific artificial cultivation of liquorice in the future.